The invention relates generally to enzymes, and specifically to solid phase enzyme kinetics screening in microcolonies of biological cells.
Demand for New Enzyme Activities
Enzymes are increasingly being used as catalysts in industry, agriculture, medicine and scientific research. Due to their substrate specificity, chemical selectivity and environmental compatibility, enzymes offer advantages for such applications as the synthesis of chirally pure pharmaceuticals, textile processing, food processing, medical diagnostics and therapy, biotransformation and bioremediation (Ogawa and Shimizu, 1999; Marrs et al., 1999; Bull et al., 1999). Enzymes are proving to be superior to traditional chemical processes for modifying high molecular weight polymers (Akkara et al., 1999). A review of enzymes as biocatalysts and their incorporation into industrial processes can be found in Uhlig et a. (1998).
Although many wild-type enzymes (i.e., those whose amino acid sequences are the same as those found in naturally occurring organisms) can be used without any modification, there are many instances wherein the physical properties of an enzyme or its chemical activity are not compatible with a desired application. Novel physical properties which might be desirable could include, for example, thermal stability, resistance to non-aqueous solvents, salt, metals, inhibitors, proteases, extremes of pH and the like. Reducing the size of the enzyme, abolishing its dependence on cofactors or other proteins, improving its expression in the host strain and other similar changes might also be desirable for a particular application. Improved chemical activities might include, for example, enhanced catalytic rate, substrate affinity and specificity, regioselectivity, enantioselectivity, reduced product inhibition, or an altered pH-activity profile. In addition, it may be desirable to alter the properties of one or more enzymes that function together as part of a metabolic pathway (Nielsen, 1998; Hutchinson, 1998; Jacobsen and Khosla, 1998).
Mutagenesis Techniques for Improving Enzymes
Mutations that encode amino acid changes can be useful for generating novel enzyme activities. The genes can be obtained using any method known to one of skill in the art, e.g. by isolating clones from a genomic library of a given organism, by polymerase chain reaction (PCR) amplification from a source of genomic DNA or mRNA, or from a library of expression clones from a heterogeneous mixture of DNA from uncultivated environmental microbes (U.S. Pat. No. 5,958,672). There are numerous methods that are well known to those skilled in the art for mutating the genes encoding enzymes and other non-catalytic proteins and peptides. These methods include both rational (e.g., creating point mutants or groups of point mutants by site-directed mutagenesis) and stochastic (e.g., random mutagenesis, combinatorial mutagenesis and recombination) techniques. One embodiment of rational design, termed protein design automation, uses an algorithm to objectively predict protein sequences likely to achieve a desired fold. Stabilized protein sequences can be designed by combining potential functions that model a protein sequence""s compatibility with a desired structure, and fast optimization tools that can search the enormous, number of sequence possibilities that occur in sequence space (Dahiyat and Mayo, 1996; Dahiyat et al., 1997; Dahiyat, 1999; Pat. Application No. WO 98/47089). In one embodiment, this method of quantitative protein design and automation can be used to search sequence space to pre-screen enormous sequence libraries, thereby reducing the size of the library that must be experimentally screened. Stochastic methods include, for example, chemical mutagenesis (Singer and Kusmierek, 1982), recursive ensemble mutagenesis (Arkin and Youvan, 1992; Delagrave et al., 1993), exponential ensemble mutagenesis (Delagrave and Youvan, 1993), sequential random mutagenesis (Chen and Arnold, 1991; 1993), DNA shuffling (Stemmer, 1994a,b) and the like. These techniques may be used individually or in combination to produce mutations. However, because the mutations are produced randomly or semi-randomly, a selection or screen must be used to identify which clones contain desirable mutations.
The stochastic methods can be used to generate an ensemble or library of mutated genes that have been cloned into plasmids or other vectors, wherein each copy of the gene may have a different sequence. The mutagenized library may contain up to 107 or more different members, and is therefore often. referred to as a high-complexity library. Generating a high complexity library is essential if a desirable mutation or class of mutations is represented at. a very low frequency within the population. When all or part of the library containing the mutated genes is expressed in an appropriate host organism (e.g., E. coil), the expressed enzyme activity can be assayed, and the clones containing the desired activity can be purified. DNA encoding the desired enzyme or protein can then be isolated from this expression library and sequenced. By repeating the steps of mutagenesis and screening, novel enzymes and other proteins can be artificially created. This iterative process is known as directed evolution. The genes of interest do not necessarily have to be expressed on plasmids. They can also be expressed following integration into the host chromosome or as a result of mutating the chromosomal copy of a gene. Note also that high complexity expression libraries can be created without mutagenesis. This can be done by cloning and expressing DNA from a source that already contains a large number of different sequences, such as highly heterogeneous genomic DNA from a mixture of environmental microbes.
Activity Screening of Expression Libraries
Screening for the desired biological activity can be done by contacting the host cells expressing the enzyme with a chromogenic or fluorogenic compound that is appropriate for the enzyme reaction and monitoring the formation of color in the cells or their surroundings. In the solid-phase assays described in U.S. Pat. No. 5,914,245, these compounds are referred to as optical signal substrates because they produce a measurable change in absorbance, reflectance, fluorescence or luminescence when they come in contact with active enzyme or with a product of the enzymatic reaction. These substrates can be obtained from a variety of suppliers, including Molecular Probes (Eugene, Oreg.), Sigma-Aldrich (St. Louis, Mo.), Biosynth (Naperville, Ill.), Research Organics (Cleveland, Ohio), CarboMer (Westborough, Mass.) and Megazyme (Wicklow, Ireland). For liquid phase assays, activity screening can be done, for example, by picking individual colonies from a growth plate, transferring each colony to an appropriate buffer solution in the well of a microplate, adding a chromogenic or fluorogenic substrate, and monitoring the change in absorbance or fluorescence with a microplate reader. An example of this method can be found in Moore and Arnold (1996). For solid phase assays, which are described in U.S. Pat. No. 5,914,245, the colonies can be maintained on a substantially continuous base, such as a microporous polymeric membrane filter, during the steps of deposition, growth, induction, lysis and assay. Microporous membranes contain numerous randomly distributed pores having a diameter of less than about 20 micrometers, and typically less than about 1 micrometer.
U.S. Pat. No. 5,914,245 also describes why it is advantageous to use microcolonies instead of colonies for screening biological activity. A microcolony is a clump of cells that are clonally derived from a single parent cell. A microcolony differs from a colony in that a colony is visible to the naked eye, whereas a microcolony need not be visible. A microcolony can be composed of any biological cells, including those from the domains Archaea, Bacteria or Eucarya. Note, however, that in addition to microcolonies, the solid-phase technique can also be used to screen phage plaques. Advantages of solid phase screening include ease of handling and reduced substrate usage. Advantages of using microcolonies versus colonies for this purpose include improved uniformity of color development across each colony and higher overall throughput per assay.
Screening Using the MicroColonyImager (MCI)
KAIROS has developed the MicroColonyImager (MCI) in conjunction with a colorimetric solid phase assay system to screen libraries expressing mutagenized enzymes or other proteins for enhanced biological activity. Using microcolonies grown at a nearly confluent density of 50,000 colonies per assay disk, this high-throughput system: (a) obtains high-resolution spectral data and makes kinetic measurements on each microcolony on the assay disk, (b) requires less than 100 nanoliters of substrate per assayed microcolony, and (c) detects as little as a two-fold difference in enzyme rates among microcolonies. The instrument can be used in conjunction with various chromogenic, fluorogenic, lumigenic and fluorescence resonance energy transfer (FRET) substrates to measure biological activity. We have previously demonstrated, for example, that the MCI technology can be used in multiplexed assays to distinguish microcolonies that show a preference for hydrolyzing the substrate Red-gal (6-Chloro-3-indolyl-Beta-D-galactopyranoside) over its epimer, X-glu (5-Bromo-4-chloro-3-indolyl-Beta-D-glucopyranoside). A description of the MCI device and methods for employing the device for solid-phase screening, as well as examples of the MCI graphical user interface can be found in U.S. Pat. No. 5,914,245.
As the need for large, highly complex mutagenized libraries increases, there is a concomitant need to develop better solid-phase screening technology to find desirable mutants. Accordingly, new methods and hardware are provided herein for accomplishing this end.
The present invention provides assay methods and membrane filter materials as well as hardware modifications for performing solid-phase enzyme screening in microcolonies of biological cells. These improvements make it easier to assay a wide variety of enzymes, provide assay membranes that have easier handling characteristics and chemical resistance, increase the overall throughput of the device, permit better temperature control of the reaction chamber and reduce the optical light path for the illumination system.
The present invention provides a means to improve methods for imaging and analyzing microcolonies of cells, for performing solid-phase directed evolution enzyme screening, and for performing solid-phase enzyme discovery screening. These methods are improved by incorporating additional solid-phase assays, including direct and indirect assays using indicators, coupled assays using indicators and indicator enzymes, coupled assays wherein the enzyme being screened produces a reaction product that is not a substrate for the indicator enzyme, inhibitor desensitization assays, and assays for screening enzymes that synthesize, modify or depolymerize high molecular weight substrates. In particular, additional types of optical signal substrates are described for use in microcolony screening, as well as methods for incorporating them into high-throughput solid-phase assays. Many of these assays are useful for screening enzymes that transform high-molecular weight substrates. Such enzymes are difficult to assay without using the solid-phase format. The improvements described in the present invention permit the user to more easily screen these enzyme activities in a high-density solid-phase format.
The present invention also incorporates additional polymer compositions for the substantially continuous base or membrane which is used to hold the microcolonies. Additional materials for the wick, which supplies optical signal substrate to the microcolonies, are also described. Microporous polyester membranes, for example, were found to provide better membrane handling characteristics and resistance to warping, particularly in the presence of non-aqueous solvents. There was also a need to expand the size of the substantially continuous base for each assay so that more microcolonies can be simultaneously measured. Increasing the size of the assay membrane to occupy more of the viewable area of the device improves the overall throughput for each assay. The original paper wick can be replaced by a hydrated gel comprising a polymer, such as agarose or polyacrylamide, in addition to the substrate. This provides highly uniform contact between the microcolonies and the substrate.
The invention further provides improvements to the illumination systems and temperature control of the MicroColonyImager. The light path and overall size of the instrument can be reduced by replacing the integrating sphere with a glass diffuser. More uniform heating of the sample compartment can be achieved by incorporating an electrical heater and a temperature feedback circuit. The present invention also describes additional types of detectors that can be used with the MCI system.